Reagents were from ThermoFisher (Invitrogen) unless stated otherwise.
iPSC culture and transduction with GT vectors
Until the start of experiments, p47 deficient CGD iPSC and healthy control hES cells HUES2 were cultured in 6 well plates in co-culture with mitomycin C inactivated mouse embryonic fibroblast (MEF) feeder cells in hES medium 2 ml/well, with daily half medium changes and passage to new plates by manual passage every 5 days. hES medium consisted of KO DMEM, 20% KO serum replacement, 2 mM Gluta-MAX, 50 nM 2-mercaptoethanol, 10 ng/ml bFGF, 100 units/ml penicillin, and 100 μg/ml streptomycin (PAA Laboratories GmbH).
For viral transduction, iPSC were transferred enzymatically with TrypLEexpress to a 48 well tissue culture plate (50.000 cells/well) and cultured feeder free on matrigel with mTeSR complete medium (Stem Cell Technologies Inc) with daily full medium change. The following day iPSC were transduced with lentiviral GT vectors. For transduction viral supernatants were diluted in mTeSR medium (Stem Cell Technologies Inc) supplemented with 4 ng/ml polybren to infect iPSC with MOI 2. Viral infectious particles were centrifuged for 45 min at 1200 g, 15°C onto the iPSC growing on matrigel followed by incubation at 37°C for 4 h. Then the medium was exchanged completely. 2 days after transduction iPSC were transferred enzymatically back onto MEFs and were further cultured in hES medium and enzymatically transferred to new plates every 3–6 days.
Differentation of iPSC to neutrophils
The neutrophil differentiation protocol described here was not published previously, but was informed by the following myeloid differentiation protocols. At day 13 after iPSC transduction one well of a 6 well plate with densely grown iPSC colonies on MEF feeder cells was used to setup embryoid bodies (EBs). Using a 24G syringe needle a grid with 100 patches was scratched onto the iPSC containing well of a 6 well plate and the patches were lifted by a cell scraper. The patches from each well were transferred into one well of a 6 well ultra-low adherence plate (Corning) in 3 ml/well hES culture medium and cultured for 6 days, with a half medium change after 2 days. iPSC patches were kept in intermediate neutrophil differentiation medium for 6 days to form EBs, with half medium change after 2 days. Intermediate neutrophil medium consisted of IMDM (PAA Laboratories GmbH, 15% FCS (Biosera), supplemented with IGFII, 20 ng/ml, VEGF 20 ng/ml, SCF100 ng/ml, FLT-3L 10 ng/ml, TPO 50 ng/ml and G-CSF 100 ng/ml, 2 mM glutamax, 100 units/ml penicillin, and 100 μg/ml streptomycin and 0.055 mM β-mercaptoethanol. For each transfected cell line 10–15 EBs were transferred to one well of a normal tissue culture 6 well plate and maintained for another 19 days in 4 ml/well of intermediate neutrophil differentiation medium with half medium changes every 5th day. Then culture conditions were changed to terminal neutrophil differentiation medium for the whole factory containing G-CSF as the only growth factor to improve differentiation of neutrophils. Terminal neutrophil differentiation medium consisted of IMDM (PAA Laboratories GmbH), 15% FCS (Biosera), supplemented with G-CSF 100 ng/ml, 2 mM Glutamax , 100 U/ml penicillin, and 100 μg/ml streptomycin and 0.055 mM β-mercaptoethanol. Emerging neutrophils were harvested every 5 days and medium was replaced.
p47phox transgene expression analysis
Transgene expression was analyzed on a FACS Calibur (BD). Cells were incubated with surface antibodies for 30 min and washed twice with PBS. Then intracellular staining was performed with Fix/Perm kit (BD) according to manufacturer's description. Transgene expression was detected with anti p47phox antibody (BD, clone 1), which was APC labeled by BD services. To identify iPSC, no surface staining was performed, instead, anti hOCT3/4 IgG2B rat monoclonal primary anti body (R&D, Cat-No: MAB1759) was used together with p47-APC antibody, then cells were washed twice with perm wash buffer followed by a staining with Alexa488 conjugated goat anti rat IgG H+L (Cat-No: A11006) antibody for 30 min. Finally iPSC were washed twice with perm wash buffer. After staining the cells were fixed with PBS containing 2% formaldehyde and stored light protected at 4°C until measurement.
Macrophages were identified with CD14-FITC (BD, Cat-No: 345784), and neutrophils with CD15-FITC (BD, Cat-No: 347423 antibody.
The DHR assay was performed as previously described.
The neutrophil yield was stained with CD15-APC antibody (BD, Cat-No:551376) for 15 min on ice. After washing cells were incubated 15 min with HBSS+Ca+Mg (PAA Laboratories GmbH, Pasching, Austria), 0.5% human serum albumin, 0.725 fM DHR (Sigma, St. Luis, USA), 1 U/ml catalase (Sigma, St. Luis, USA, Cat No: C 9322). Then cell suspension was divided on 2 FACS tubes. One tube was left unstimulated, the other tube was stimulated with 0.4 ng/ml PMA (Sigma, St. Luis, USA). Both tubes were incubated at 37°C for 15 min and measured immediately after.
SFFV promoter methylation analysis
Promoter methylation analysis was performed by bisulfite conversion as described previously for the clinical trial of X-CGD. In brief, isolated DNA was bisulfite converted using the Epi Tect kit (Qiagen AG). Bisulfite converted DNA was amplified by PCR and cloned into TOPO vector for sequencing.
The PCR of SFFV promoter methylation analysis amplified the enhancer and CpG island region of the SFFV promoter as well as the first 7 CpGs of the p47phox transgene and the PCR was carried out using the primer “SFFVfwdbisulfite” 5’-AATTA AGAAT AGAGA AGTTT AGATT AAGGG-3’ and primer “METH_SFFV_rev” 5'-TCCTA CCACT TCACCA AAAAC ATATAC-3’ TOPO vector subcloned PCR products were sequenced with M13forward and M13reverse standard primers.
Maturity of neutrophils and iPSC macrophages was monitored with an eosin/methylene blue staining as described by. About 2×104 cells were centrifuged on an X-tra® adhesive slide (Surgipath, Canada) using a cytospin device. The sample was then fixed for 1 min in methanol, stained for 30 s with eosin solution directly followed by staining with methylene blue solution for 30 s. Then the slides were washed twice in distilled water for 30 s and placed in a drying oven until water was evaporated. Images were recorded with an EVOSxl core inverted microscope (AMG).